Dragana Matić

Cadmium-induced changes of gypsy moth larval mass and protease activity

Cadmium uptake takes place mainly through food. Lymantria dispar larvae were exposed to dietary cadmium in concentrations of 10 and 30μg Cd/g dry food (NOEC, no-observed-effect and LOEC, lowest-observed-effect concentration, respectively) for acute and chronic treatment and recovery. We established that metal contamination decreased mass only during the chronic treatment at 30μg Cd/dry food with no recovery on removal of cadmium for 3days. Significant reduction of protease activity was detected at LOEC after the acute and chronic treatments. Protease showed enhanced plasticity with regard to the fitness trait (mass) during environmental stress and the higher cadmium load, when it changed. The statistically significant higher index of phenotypic plasticity for protease correlated with lower variability. Protease isoforms at the same cadmium treatments differed between genotypes, while some protease isoforms from one egg-mass differed between cadmium treatments. Owing to the low sensitivity and plasticity of mass change during exposure to cadmium, as well as its small influence, we concluded that larval mass is not a good indicator of cadmium presence in food. We suggest that proteases, with further research, might be a suitable indicator of dietary cadmium contamination, as well as nutriment utilization during heavy metal stress.

Acetylcholinesterase (AChE) and heat shock proteins (Hsp70) of gypsy moth (Lymantria dispar L.) larvae in response to long-term fluoranthene exposure.

Polycyclic aromatic hydrocarbons (PAHs) may affect biochemical and physiological processes in living organisms, thus impairing fitness related traits and influencing their populations. This imposes the need for providing early-warning signals of pollution. Our study aimed to examine changes in the activity of acetylcholinesterase (AChE) and the concentration of heat shock proteins (Hsp70) in homogenates of brain tissues of fifth instar gypsy moth (Lymantria dispar L.) larvae, exposed to the ubiquitous PAH, fluoranthene, supplemented to the rearing diet. Significantly increased activity of AChE in larvae fed on the diets with high fluoranthene concentrations suggests the necessity for elucidation of the role of AChE in these insects when exposed to PAH pollution. Significant induction of Hsp70 in gypsy moth larvae reared on the diets containing low fluoranthene concentrations, indicate that changes in the level of Hsp70 might be useful as an indicator of pollution in this widespread forest species.

Glutathione S-transferase in the midgut tissue of gypsy moth (Lymantria dispar) caterpillars exposed to dietary cadmium.

Activity of glutathione S-transferase (GST) in midgut of gypsy moth caterpillars exposed to 10 and 30μg Cd/g dry food was examined. Based on the enzyme reaction through conjugation with glutathione, overall activity remained unaltered after acute and chronic treatment. No-observed-effect-concentration (10μg Cd/g dry food) significantly increased activity only after 3-day recovery following cadmium administration. Almost all comparisons of the indices of phenotypic plasticity revealed statistically significant differences. Despite the facts that GST has important role in xenobiotic biotransformation, our results indicate that this enzyme in insect midgut does not represent the key factor in cadmium detoxification.

Ghrelin effect on nutritional indices, midgut and fat body of Lymantria dispar L. (Lymantriidae)

Ghrelin is a 28-amino acid peptide that has significant effects on appetite and growth in humans and animals. The aim of this study was to examine 4th instar larvae of the pest insect Lymantria dispar L. after ghrelin treatment. Parameters included changes in nutritional indices (efficiency of conversion of ingested food, efficiency of conversion of digested food, approximate digestibility); midgut and fat body mass; total proteases, trypsin and leucine aminopeptidase activities in the midgut; number, height and width of columnar and goblet cells and their nuclei in the midgut epithelium and detection of ghrelin-like immunoreactivity in the midgut tissue. Four subpicomolar injections of ghrelin (0.3pmol) or physiological saline (control) were applied every 24h. The nutritional indices were higher in the ghrelin treated than in the control group. Ghrelin treatment was also associated with elevation of midgut mass, induced digestive enzyme activities, increased fat body mass and morphometric changes in columnar and goblet cells. This is the first report of the presence of ghrelin-like hormone in endocrine cells of an insect midgut. Such information provides additional evidence for application of this relatively simple model system in the future studies of the mechanisms underlying of digestion and energy balance in more complex organisms.

Influence of dietary cadmium exposure on fitness traits and its accumulation (with an overview on trace elements) in Lymantria dispar larvae

Bioaccumulation and excretion of heavy metals in insects is only partially clarified. We have investigated cadmium accumulation in the feces, head and integument of Lymantria dispar (Lepidoptera: Lymantriidae) larvae exposed to chronic dietary intake of cadmium. The aim of the experiment was to establish modalities of metal accumulation, primarily cadmium, as well as changes in fitness traits in two insect populations receiving 50 or 100μgCd/g dry food. The egg-masses originated from two localities: a protected nature reserve (unpolluted population) and an area near a busy highway (polluted population) in Serbia. At both added dietary levels cadmium concentration was highest in feces. Small alterations in metal concentrations after both cadmium treatments were detected in the integument. It was established that irrespective of population origin, the modality of decline of larval mass and relative growth rate (RGR) was similar. Concentrations of Cu and Zn in the integument were approximately the same regardless of dietary cadmium intake. However, cadmium accumulation in the examined tissues, as well as variability of fitness traits depended on population origin and cadmium concentration. In larvae from both populations not given contaminated food the head was the organ with the greatest accumulation of trace elements. Our results reveal how invasive phytophagous insects cope with high metal concentrations in their food by body mass (RGR) reduction and energy allocation towards processes that enable accumulation of cadmium and other trace elements in different tissues.

Response of α-glucosidase in gypsy moth larvae to acute and chronic dietary cadmium.

We investigated the effects of acute and chronic treatments with cadmium at 10 μg Cd/g dry food and 30 μg Cd/g dry food on α-glucosidase activity of the 4th instar larvae of Lymantria dispar (Lepidoptera: Lymantriidae) as well as subsequent recovery. Enzyme inhibition was recorded during acute exposure to 30 μg Cd/g dry food and during chronic treatment at the lower metal concentration. After three days recovery from 10 μg Cd/g dry food, the α-glucosidase activity returned to the control level. One-way ANOVA showed that cadmium significantly influenced the activity of α-glucosidase during all treatments. The index of phenotypic plasticity was higher during chronic treatment at 10 μg Cd/g dry food than at 30 μg Cd/g as well as during the recovery. We detected four glucosidase isoforms by NATIVE PAGE. The activities and expressions of the isoforms depended on both larval genotype and cadmium treatment.

The specific response of gypsy moth A1 neurosecretory neurons to different environmental stressors

Changes in the number and morphometric parameters of A1 neurosecretory neurons (nsn) were analyzed in Lymantria dispar 4th instar caterpillars, exposed for 3 days to different stressors: cadmium, high temperature and tannic acid. The relative cytoplasm density of A1 nsn was also estimated. Caterpillars reared on a diet supplemented with cadmium exhibited increased size of A1 nuclei (10 and 250 μg Cd per g of dry food weight), increased number of nucleolii in nuclei and raised relative cytoplasm density in all experimental groups. Cadmium obviously induces intensive synthetic activity in A1 nsn. The second stressor was high environmental temperature of 35°C. Decrease of all analyzed morphometric parameters suggests that acute exposure of 4th instar caterpillars to 35°C, as well as 12 h recovery at optimal temperature of 23°C, reduced the activity of A1 nsn. Tannic acid was added to the artificial diet in the following concentrations: 1%, 2.5% and 5%. All estimated morphological parameters did not change after addition 1 and 2.5% of tannic acid. After addition of 5% of tannic acid, the activity of A1 nsn declined.

Tannic acid modulates the activity of bombyxin- and PTTH-producing neurons in Lymantria dispar L. (Insecta: Lepidoptera) caterpillars

Abstract Lymantria dispar caterpillars were exposed for 3 days to tannic acid, added to the artificial diet in the following concentrations: 1, 2.5 and 5%. Changes in the morphometric parameters of medial A2 bombyxin immunopositive and dorsolateral L2` prothoracicotropic (PTTH) immunopositive neurosecretory neurons (nsn; sizes of six nsn, the size of their nuclei, and quantification of cytoplasm density) were analysed upon caterpillars’ exposure to this allelochemical. Protein band density, obtained by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) electrophoresis, from regions of bombyxin and PTTH molecular mass (Mr) were estimated. A2 nsn decreased in size, while the size of their nuclei did not change significantly. Relative cytoplasm density and relative band density from the bombyxin Mr region (Mr 4–5 kD) were decreased upon exposure to 1% and 2.5% tannic acid, while the addition of 5% tannic acid to artificial diet increased all of these parameters. Results suggested that lower concentrations of tannic acid stimulate the release of neurosecretory material from A2 nsn, most probably bombyxin-like protein. High tannic acid concentration (5%) probably allocates resources towards induction of other stress protective components instead of bombyxin-mediated metabolic stress response mechanisms. In L2` nsn, the size of nsn, nuclei and the relative cytoplasm density were increased, and relative band density from the region of big PTTH form (Mr 11–15 kD) was higher in all treatments in comparison to control. Results indicate that PTTH is synthesised and stored in the cytoplasm of L2` nsn.